1. Field of the Invention
The present invention relates to the microbiological industry, and specifically to a method for producing an L-amino acid by fermentation of a bacterium of the family Enterobacteriaceae that has been modified to attenuate expression of a phosphate transporter-encoding gene.
2. Brief Description of the Related Art
Conventionally, L-amino acids are industrially produced by fermentation methods utilizing strains of microorganisms obtained from natural sources, or mutants thereof. Typically, the microorganisms are modified to enhance production yields of L-amino acids.
Many techniques to enhance L-amino acid production yields have been reported, including transformation of microorganisms with recombinant DNA (see, for example, U.S. Pat. No. 4,278,765 A), and alteration of regulatory regions such as promoter, leader sequence, and/or attenuator, or others known to the person skilled in the art (see, for example, U.S. Patent Application Publication No. 20060216796 A1 and WO9615246 A1). Other techniques for enhancing production yields include increasing the activities of enzymes involved in amino acid biosynthesis and/or desensitizing the target enzymes to the feedback inhibition by the resulting L-amino acid (see, for example, WO9516042 A1, EP0685555 A1 or U.S. Pat. Nos. 4,346,170 A, 5,661,012 A, and 6,040,160).
Another method for enhancing L-amino acids production yields is to attenuate the expression of a gene or several genes which are involved in the degradation of the target L-amino acid, genes which divert the precursors of the target L-amino acid from the L-amino acid biosynthetic pathway, genes involved in the redistribution of the carbon, nitrogen, and phosphate fluxes, and genes encoding toxins, etc.
Bacteria, such as, for example, Escherichia coli (E. coli), contain two pit genes, pitA and pitB, which encode proteins that transport inorganic phosphate (Pi) across the cytoplasmic membrane of the bacteria (Harris R. M. et al., Characterization of PitA and PitB from Escherichia coli, J. Bacteriol., 2001, 183(17):5008-5014). The phosphate transporters PitA and PitB are protein homologs having an amino acid sequence identity of 81% relative to each other (Hoffer S. M. et al., Activation by gene amplification of pitB, encoding a third phosphate transporter of Escherichia coli K-12, J. Bacteriol., 2001, 183(15):4659-4663), and these transporters belong to the Inorganic Phosphate Transporter (PiT) family. A screening of the 34 completely sequenced bacterial genomes (ncbi.nlm.nih.gov/COG) revealed that some bacteria, such as, for example, Pseudomonas aeruginosa (Pseudomonadaceae), contain more than one PitA homolog.
The PitA and PitB from the prokaryotic E. coli can transport inorganic phosphate independently of each other. Such overlapping activity of inorganic phosphate transporters has been also reported for eukaryotic species such as, for example, Saccharomyces cerevisiae (Martinez P. R. et al., Physiological regulation of the derepressible phosphate transporter in Saccharomyces cerevisiae, J. Bacteriol., 1998, 180:2253-2256) and Neurospora crassa (Versaw W. K. and Metzenberg R. L., Repressible cation-phosphate symporters in Neurospora crassa, Proc. Natl. Acad. Sci. USA, 1995, 92:3884-3887). Previous studies on prokaryotes have shown that the PitA system, which can also be referred to as the Pit system, mediates the uptake of inorganic phosphate and divalent cation ions. For example, the uptake of inorganic phosphate in E. coli (Enterobacteriaceae) and Acinetobacter johnsonii (Moraxellaceae) by utilizing the Pit system is dependent on co-transport of divalent metal cation ions, such as Mg(II), Ca(II), Mn(II), or Co(II), through the formation of a soluble, neutral, metal-phosphate complex, which is the transported species (van Veen H. W. et al., Translocation of metal phosphate via the phosphate inorganic transport system of Escherichia coli, Biochemistry, 1994, 33(7):1766-1770; van Veen H. W. et al., Mechanism and energetics of the secondary phosphate-transport system of Acinetobacter-johnsonii-210A. J. Biol. Chem., 1993, 268:19377-19383). It has been also reported that PitA is involved in Zn(II)-uptake probably via formation of a ZnHPO4 complex. However, it is suggested that PitA may also play the role in Zn(II)-efflux, when the intracellular concentration of zinc ions becomes toxic (Beard S. J. et al., Evidence for the transport of zinc(II) ions via the pit phosphate transport system in Escherichia coli, FEMS Microbiol. Lett., 2000, 184:231-235).
However, no data has been previously reported that describes the effect of attenuating expression of a phosphate transporter-encoding gene on production of L-amino acids by fermentation of an L-amino acid-producing bacterium of the family Enterobacteriaceae.